小鼠睾丸体外生精小管模型构建及Rho/ROCK信号通路对其重构的影响  

Reconstruction of a seminiferous tubule-like structure in vitro and the effect of Rho/ROCK signaling on the reconstruction of seminiferous tubule-like structure

作　　者：孙梦迪 李春阳 刘松坡 李小明 徐睿 兰东锋 代小方 张继东 Sun Mengdi;Li Chunyang;Liu Songpo;Li Xiaoming;Xu Rui;Lan Dongfeng;Dai Xiaofang;Zhang Jidong(Department of Immunology,Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Genetics,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学免疫学教研室,贵州遵义563099 [2]遵义医科大学遗传学教研室,贵州遵义563099

出　　处：《遵义医科大学学报》2022年第2期147-152,共6页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31960156,31660338);省部共建协同创新中心项目(NO:教科技厅函[2020]39)。

摘　　要：目的构建小鼠睾丸体外生精小管模型,探讨Rho/ROCK信号通路在生精小管重构过程中的作用。方法取新生10日龄小鼠睾丸,分离纯化睾丸支持细胞(Sertoli cells,SCs),培养3 d后利用免疫荧光染色技术检测分离细胞GATA结合蛋白4(GATA Binding protein 4,GATA4)表达情况,鉴定SCs纯度;以RPMI 1640为阴性对照组(cont组),人工合成血清替代物(KnockOut^(TM) Serum replacement,KSR)和FBS为实验组,倒置显微镜下观察原代SCs生精小管形成情况,建立生精小管体外培养模型;分别以RPMI 1640培养基组和KSR组为阴性对照和阳性对照组,以KSR+Y27632(K+Y)、缓激肽(Bradykinin,BK)为实验组,探讨Rho/ROCK信号通路在生精小管形成过程中的作用。结果(1)免疫荧光染色结果显示在分离得到的细胞中GATA4^(+)DAPI^(+)SCs占90%以上;(2)倒置显微镜下观察到与cont组相比,随着培养天数的增加,KSR组有大量管状结构形成,而FBS组只观察到细胞生长,并未有管状结构形成;(3)添加ROCK分子抑制剂Y27632后镜下观察到的管状结构被明显抑制,而当添加Rho/ROCK信号通路激动剂BK后,我们观察到类似于KSR组管状结构的形成。结论Rho/ROCK信号通路参与小鼠睾丸体外生精小管结构的形成。Objective To construct a seminiferous tubule model in vitro and observe the role of Rho/ROCK signaling in the reconstruction of seminiferous tubule.Methods The 10-dpp mouse(C57BL/6)testis were digested to obtain Sertoli cell suspension.Immunocytochemistry to identify Sertoli cells(SCs)with specific antibody against GATA4(GATA Binding Protein 4)after 3 days of cultivation.Then the Sertoli cell suspension were cultured in RPMI 1640,KSR and FBS medium respectively to construct the seminiferous tubule model in 2D culture.Finally,using the ROCK inhibitor Y27632 and the Rho agonist BK to explore the role of Rho/ROCK signaling pathway in the formation of seminiferous tubule-like stucture.Results(1)Immunofluorescence staining showed that GATA4+DAPI+SCs accounted for more than 90%;(2)Compared with cont.group,the addition of KSR promoted the formation of tubular structures,while cell growth was observed in FBS medium without tubular structures and noticed cell elongation only;(3)More intriguingly,the tubular structures in KSR were significantly inhibited by the addition of ROCK inhibitor Y27632.Contrastedly,the Rho GTPase agonist BK could promote tubular structures formed.Conclusion Rho/ROCK signaling pathway is involved in the formation of 2D seminiferous tubules.

关 键 词：生精小管形成 支持细胞 RHO/ROCK信号通路 

分 类 号：R392.9[医药卫生—免疫学]